Use of Quorum Quenching (QQ) Methodologies for the Treatment of Bacterial Infections
A number of pathogenic bacteria coordinate their infectious activity by means of intercellular communication processes referred to as Quorum Sensing (QS) (Otero Casal et al., 2004). These processes consist of the release into the medium of small signal molecules that allow the bacteria to quantify the presence of other bacteria by means of specific sensors, acting in a coordinated manner once the threshold concentration of the signal indicating the existence of quorum is reached. QS controls the production of virulence factors, such as exoenzymes or pigments, of a number of human, animal and plant pathogens (Whitehead et al., 2001; Fuqua and Greenberg, 2002; Bassler and Losick, 2006; Williams et al., 2007), including important nosocomial pathogens such as Pseudomonas aeruginosa. 
Since the virulence of a number of bacterial pathogens depends on QS processes, the inactivation or interception of communication mediated by quorum signals, a strategy generically referred to as quorum sensing inhibition or Quorum Quenching (QQ), is an alternative to the use of antibiotics for the treatment and prevention of bacterial infections. QQ processes have enormous potential in the pharmaceutical and biotechnological fields since it is a virulence biocontrol mechanism that prevents bacteria from launching their attack, making them more sensitive to the host's defenses and making it easier for them to be eliminated with greater efficiency. A key point of QQ strategies is that they do not affect bacteria viability, but rather only interfere in the expression of virulence factors, preventing selective pressure on them, and therefore not generating resistances.
QQ mechanisms are considered to be the new antipathogenic agents, making them an important object of study, since one of the possible uses thereof is the control of both plant and animal pathogens, including human pathogens, acting through QS processes. The QQ process has a lower probability of resistance selection and higher specificity, affecting only the recipient bacteria. Furthermore, the use of QQ strategies accompanied by antibiotics could give rise to the control of multiresistant pathogens, such as P. aeruginosa. In aquaculture, the use thereof as antibiotic substitutes is an alternative with great potential due to the legal restrictions on the use of antibiotics in this field, as well as in other animal health fields. In turn, QS and QQ processes take on real importance in the marine environment due to their ecological implications therein, and particularly in the field of aquaculture, because QS processes mediated by N-acyl-homoserine lactones (AHLs) or type 2 autoinducers (AI-2) are very widely spread in marine pathogens, as in the case of Vibrio harveyi, V. anguillarum, V. salmonicida, V. vulnificus, Aeromonas salmonicida, A. hydrophila, Yersinia ruckeri, Edwarsiella tarda and Tenacibaculum maritimum (Freeman and Bassler, 1999; Swift et al., 1999; Croxatto et al., 2002; Buch et al., 2003; Morohoshi et al., 2004; Bruhn et al., 2005; Romero et al., 2010).
A process controlled by QS phenomena to be highlighted is biofilm formation, which has a huge economic and clinical impact. It is known that many of these biofilms are closely related to human infectious processes. The mechanisms whereby biofilm produces disease symptoms are still not completely established, although it has been suggested that biofilm bacteria can produce endotoxins, groups of bacteria can be released into the bloodstream, they become resistant to the phagocytic action of immune system cells, and, in addition, are a niche for the occurrence of bacteria resistant to antibiotic treatments. This last aspect can be particularly relevant given that resistant bacteria generated in a biofilm could spread from patient to patient through the hands of healthcare staff. Biofilm-forming pathogens are very resistant to antibiotics and can adhere to foods or to substances in contact with such foods, causing both hygiene problems and possible food-borne diseases and, finally, large economic losses. Furthermore, they are often located on the surface of the medical implants or in devices inserted in the organism. They can also be formed in areas of the body that are exposed to the air; particularly, in wounds and in the pleura. In this sense, Pseudomonas aeruginosa is one of the most infective and problematic bacteria since it forms biofilms that are hard to treat with conventional antibiotics. In patients with cystic fibrosis, it colonizes the lungs causing hard-to-treat and, often, finally fatal infections. It is also particularly interesting in patients with chronic wounds or burns. In addition, Staphylococcus aureus and Staphylococcus epidermidis are currently classified as the main causes of nosocomial infections. This situation is favored by the fact that these species live both in mucous membranes and in the skin of human beings which allows them to enter the patient's bloodstream through surgical wounds by means of direct or indirect contact with healthcare staff, with a contaminated object or even with another patient. These species form biofilms that colonize catheters, drains and implants, favoring the contamination and antibiotic resistance. Furthermore, one of the most complex and most clinically relevant biological biofilms is dental plaque, the formation of which is caused by, among other pathogens in the oral cavity, Streptococcus mutans, which also forms biofilms. Additionally, biofilms contribute to biological surface contamination, mechanical blockage in conduits, drinkable water distribution systems, air conditioning systems, fire protection systems, etc. Finally, it must be added that they favor biofouling formation as they are the basis for the growth of other higher organisms on submerged surfaces, being a serious economic problem in the aquaculture and marine transport sectors.
Therefore, there is a need to develop new antibacterial agents that increase the arsenal of remedies for controlling bacterial infections.
Marine bacterium Tenacibaculum sp. strain 20J (CECT 7426) has high QQ capacity.
Tenacibaculum sp. 20J (CECT 7426): A Strain with High QQ Capacity
Marine bacterium Tenacibaculum sp. strain 20J (CECT 7426) is characterized by high AHL signal degradation capacity, much greater than other bacteria with QQ activity that have been isolated from soil (Romero et al., 2012). This is the isolate obtained from a sample of marine fish farming tank sediment, in TSA-I medium (Romero, 2010). The partial sequence of the 16S ribosomal RNA gene of the strain has a percentage of identity of 99% with the pathogenic marine bacterium type strain Tenacibaculum discolor DSM 18842/NCIMB 14278T, belonging to the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), which causes the bacterial infection known as tenacibaculosis/flexibacteriosis, or gliding bacterial disease (Piñeiro-Vidal et al., 2008). A distinctive feature of Tenacibaculum sp. strain 20J (CECT 7426) is that it is capable of growing in media lacking marine salts (TSA-1), so if they are strictly applied, the taxonomic characters established for the genus Tenacibaculum would exclude strain 20J from the T. discolor species (Piñeiro Vidal et al., 2008). Therefore, the terminology Tenacibaculum sp. strain 20J or simply “strain 20J” will be used throughout this text to describe this species. Living cells and cell extracts of strain 20J degrade the entire size range of known AHLs, with or without oxo-substitutions, so the activity thereof is highly unspecific (Romero, 2010). It is a fast-growing strain that can be cultured both in marine environments and in terrestrial environments.
Preliminary studies performed with strain 20J indicate that the main location of its QQ activity is linked with cells, although it is also observed in filtered supernatants (ES 2342807 B2). Furthermore, its activity is constitutive, as it does not require prior exposure to signals for expression. As it presents QS intercepting activity both in living cells and in purified cell extracts (designated as CCEs in ES2342807 B2), it allows the use of both types of presentations for biotechnological use. In addition to having demonstrated its wide spectrum of QQ activity, the comparison of its activity with the data available in the literature confirms its potential because this strain degrades AHLs at a rate that is up to 24 times greater than the marine bacterial consortia selected for their QQ activity (Cam et al., 2009). Comparison of the activity of purified cell extracts of strain 20J with the same extracts of Bacillus thuringiensis strain CECT 197 clearly showed greater activity of the purified cell extracts of strain 20J, especially in the case of degrading N-hexanoyl-L-homoserine lactone (C6-HSL): whereas minimum active concentration (MAC) for completely degrading a 10 μM solution of C6-HSL in 24 hours is 10 μg of protein extract per ml for strain 20J, a concentration 3 orders of magnitude greater of Bacillus thuringiensis ATCC10792 cell extract, 10,820 μg of protein/ml, is needed to carry out the same degradation (unpublished data). As a result of all its features, strain 20J is a promising candidate for the control of pathogens related to human health, aquaculture, animals and plants or in the inhibition of biofilm formation (Romero, 2010), and therefore these uses have been protected by means of a patent (ES 2342807 B2).
Nevertheless, it has not been possible to identify up until now the gene responsible for its QQ activity or for its degradation activity of QS signals, particularly AHL signals. The identification and characterization of said gene would allow readily providing important amounts of the product responsible for said activity, which would facilitate and generalize the use thereof in any type of uses in which control of QS signal-producing bacterial populations is needed.
The efforts made up until now were unsuccessful in the attempt to clone the gene or genes responsible for QQ activity on QS signals, particularly AHLs, by means of techniques based on polymerase chain reaction (PCR) with degenerate primers, designed for the conserved sequences of lactonases and acylases described in the literature or by means of the construction of a genomic library in the plasmid that was used to transform the AHL-producing strain, P. aeruginosa PAOI, carrier of the QSIS pMH655 plasmid (Rasmussen et al., 2005), which was designed for detection of QQ, because the strain cannot survive in the presence of saccharose and AHL. Specifically, none of said methodologies resulted in detection of the genes responsible for QQ activity of strain 20J (unpublished data), which is indicative of the existence of important differences between the enzyme with QQ activity of strain 20J and the enzymes with QQ activity described up until now.